[unreadable] Recombination plays an essential mechanical role in meiotic chromosome segregation and defective recombination is linked to infertility, miscarriage and genetic disease in humans. The long term goal is to understand the molecular mechanism of recombination. Meiotic recombination occurs by the programmed formation and processing of DNA double-strand breaks (DSBs). DSB-ends interact sequentially with an homologous chromosome forming first a Single-End Invasion (SEI) and then a double-Holliday Junction (dHJ). The specific hypothesis is that the DSB-to-SEI and SEI-to-dHJ transitions occur via biochemically distinct processes, with unique contributions being made by the Dmc1, Rad51 and Rad52 proteins. DNA physical assays to directly monitor the chemical steps of meiotic recombination in Saccharomyces cerevisiae cells will form the cornerstone of this investigation. The Specific Aims are: 1. To characterize the in vivo roles of Dmc1 and Rad51. Indirect effects of null mutations and lack of in vivo assays to detect relevant strand-invasion products have previously limited our understanding of Dmc1 and Rad51 function. Preliminary experiments have identified conditions in which meiosis progresses efficiently when only Dmc1 or Rad51 is present. The following aspects of Dmc1-only and Rad51-only recombination will be characterized using physical, genetic and cytological assays: (a) the DNA events of recombination; (b) the gene products involved; (c) the distribution of crossovers formed; (d) the ability to promote chromosome pairing and formation of synaptonemal complex. 2. To characterize the mechanism of the SEI-to-dHJ transition. Events following SEI formation are uncharacterized but must include interaction of the second DSB-end, DNA synthesis and ligation. Preliminary evidence suggests the second DSB-end can interact by a process of single-strand-annealing. Physical assays will be used to characterize the following two aspects of the SEI-to-dHJ transition: (a) the roles of proteins implicated in the process of single-strand-annealing will be determined by analyzing a series of mutant strains; (b) the role of DNA synthesis and the involved factors will be established using chemical inhibitors of DNA synthesis and conditional alleles of replication proteins. [unreadable] [unreadable]